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Jurkovicova-Tarabova B, Stringer RN, Sevcikova Tomaskova Z, Weiss N. Electrophysiological characterization of sourced human iPSC-derived motor neurons. Channels (Austin) 2025; 19:2480713. [PMID: 40131207 PMCID: PMC11938304 DOI: 10.1080/19336950.2025.2480713] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2025] [Revised: 02/20/2025] [Accepted: 03/12/2025] [Indexed: 03/26/2025] Open
Abstract
Induced pluripotent stem cell (iPSC)-derived motor neurons provide a powerful platform for studying motor neuron diseases. These cells enable human-specific modeling of disease mechanisms and high-throughput drug screening. While commercially available iPSC-derived motor neurons offer a convenient alternative to time-intensive differentiation protocols, their electrophysiological properties and maturation require comprehensive evaluation to validate their utility for research and therapeutic applications. In this study, we characterized the electrophysiological properties of commercially available iPSC-derived motor neurons. Immunofluorescence confirmed the expression of motor neuron-specific biomarkers, indicating successful differentiation and maturation. Electrophysiological recordings revealed stable passive membrane properties, maturation-dependent improvements in action potential kinetics, and progressive increases in repetitive firing. Voltage-clamp analyses confirmed the functional expression of key ion channels, including high- and low-voltage-activated calcium channels, TTX-sensitive and TTX-insensitive sodium channels, and voltage-gated potassium channels. While the neurons exhibited hallmark features of motor neuron physiology, high input resistance, depolarized resting membrane potentials, and limited firing capacity suggest incomplete electrical maturation. Altogether, these findings underscore the potential of commercially available iPSC-derived motor neurons as a practical resource for MND research, while highlighting the need for optimized protocols to support prolonged culture and full maturation.
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Affiliation(s)
- Bohumila Jurkovicova-Tarabova
- Institute of Molecular Physiology and Genetics, Center of Biosciences, Slovak Academy of Sciences, Bratislava, Slovakia
- Department of Biology, Faculty of Education, Trnava University, Trnava, Slovakia
| | - Robin N. Stringer
- Department of Pathophysiology, Third Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Zuzana Sevcikova Tomaskova
- Institute of Molecular Physiology and Genetics, Center of Biosciences, Slovak Academy of Sciences, Bratislava, Slovakia
| | - Norbert Weiss
- Institute of Molecular Physiology and Genetics, Center of Biosciences, Slovak Academy of Sciences, Bratislava, Slovakia
- Department of Pathophysiology, Third Faculty of Medicine, Charles University, Prague, Czech Republic
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2
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Matsuzono K, Honda H, Mashiko T, Koide R, Sakashita E, Endo H, Kitamoto T, Fujimoto S. GPI-anchorless prion disease is sensitive to oxidative stress and shows potential for treatment with edaravone, based on iPS-derived neuron study. Cell Mol Life Sci 2025; 82:202. [PMID: 40372528 PMCID: PMC12081781 DOI: 10.1007/s00018-025-05698-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2025] [Revised: 03/27/2025] [Accepted: 04/04/2025] [Indexed: 05/16/2025]
Abstract
Only a few reports have generated induced pluripotent stem cells from patients with prion diseases, making it important to conduct translational studies using cells derived from individuals with prion protein (PRNP) mutations. In this study, we established induced pluripotent stem cells from a patient with a glycosylphosphatidylinositol-anchorless PRNP mutation (Y162X), which leads to abnormal deposits of prion protein in various organs. While no abnormal intracellular prion protein deposits were observed in the neurons differentiated from PRNP Y162X induced pluripotent stem cells, extracellular PrP aggregates secretions were significantly increased, and these cells were significantly more sensitive to oxidative stress compared to control cells. Utilizing this PRNP Y162X iPSC-derived neuron model, we discovered that edaravone reduced the sensitivity of PRNP Y162X cells to oxidative stress. Following this finding, we treated a PRNP Y162X patient with edaravone for two years, which successfully suppressed indicators of disease progression. Our study demonstrates that the pathology of the glycosylphosphatidylinositol-anchorless PRNP mutation is associated with oxidative stress and highlights the potential of induced pluripotent stem cell technology in identifying novel treatments for rare prion diseases.
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Affiliation(s)
- Kosuke Matsuzono
- Division of Neurology, Department of Medicine, Jichi Medical University School of Medicine, Yakushiji 3311-1, Shimotsuke, Tochigi, 329-0498, Japan.
| | - Hiroyuki Honda
- Department of Neuropathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
- Neuropathology Center, Department of Neurology, National Hospital Organization, Omuta National Hospital, Fukuoka, Japan
| | - Takafumi Mashiko
- Division of Neurology, Department of Medicine, Jichi Medical University School of Medicine, Yakushiji 3311-1, Shimotsuke, Tochigi, 329-0498, Japan
| | - Reiji Koide
- Division of Neurology, Department of Medicine, Jichi Medical University School of Medicine, Yakushiji 3311-1, Shimotsuke, Tochigi, 329-0498, Japan
| | - Eiji Sakashita
- Department of Biochemistry, Jichi Medical University, Tochigi, Japan
| | - Hitoshi Endo
- Department of Biochemistry, Jichi Medical University, Tochigi, Japan
| | - Tetsuyuki Kitamoto
- Division of CJD Science and Technology, Department of Neurological Science, Tohoku University Graduate School of Medicine, Miyagi, Japan
| | - Shigeru Fujimoto
- Division of Neurology, Department of Medicine, Jichi Medical University School of Medicine, Yakushiji 3311-1, Shimotsuke, Tochigi, 329-0498, Japan
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3
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González-Sánchez M, Ramírez-Expósito MJ, Martínez-Martos JM. Pathophysiology, Clinical Heterogeneity, and Therapeutic Advances in Amyotrophic Lateral Sclerosis: A Comprehensive Review of Molecular Mechanisms, Diagnostic Challenges, and Multidisciplinary Management Strategies. Life (Basel) 2025; 15:647. [PMID: 40283201 PMCID: PMC12029092 DOI: 10.3390/life15040647] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2025] [Revised: 03/28/2025] [Accepted: 04/10/2025] [Indexed: 04/29/2025] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the progressive degeneration of upper and lower motor neurons, leading to muscle atrophy, paralysis, and respiratory failure. This comprehensive review synthesizes the current knowledge on ALS pathophysiology, clinical heterogeneity, diagnostic frameworks, and evolving therapeutic strategies. Mechanistically, ALS arises from complex interactions between genetic mutations (e.g., in C9orf72, SOD1, TARDBP (TDP-43), and FUS) and dysregulated cellular pathways, including impaired RNA metabolism, protein misfolding, nucleocytoplasmic transport defects, and prion-like propagation of toxic aggregates. Phenotypic heterogeneity, manifesting as bulbar-, spinal-, or respiratory-onset variants, complicates its early diagnosis, which thus necessitates the rigorous application of the revised El Escorial criteria and emerging biomarkers such as neurofilament light chain. Clinically, ALS intersects with frontotemporal dementia (FTD) in up to 50% of the cases, driven by shared TDP-43 pathology and C9orf72 hexanucleotide expansions. Epidemiological studies have revealed a lifetime risk of 1:350, with male predominance (1.5:1) and peak onset between 50 and 70 years. Disease progression varies widely, with a median survival of 2-4 years post-diagnosis, underscoring the urgency for early intervention. Approved therapies, including riluzole (glutamate modulation), edaravone (antioxidant), and tofersen (antisense oligonucleotide), offer modest survival benefits, while dextromethorphan/quinidine alleviates the pseudobulbar affect. Non-pharmacological treatment advances, such as non-invasive ventilation (NIV), prolong survival by 13 months and improve quality of life, particularly in bulb-involved patients. Multidisciplinary care-integrating physical therapy, respiratory support, nutritional management, and cognitive assessments-is critical to addressing motor and non-motor symptoms (e.g., dysphagia, spasticity, sleep disturbances). Emerging therapies show promise in preclinical models. However, challenges persist in translating genetic insights into universally effective treatments. Ethical considerations, including euthanasia and end-of-life decision-making, further highlight the need for patient-centered communication and palliative strategies.
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Affiliation(s)
| | | | - José Manuel Martínez-Martos
- Experimental and Clinical Physiopathology Research Group CTS-1039, Department of Health Sciences, School of Health Sciences, University of Jaén, E23071 Jaén, Spain; (M.G.-S.); (M.J.R.-E.)
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4
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Urasawa T, Kawasaki N. Proteomic Approach Using DIA-MS Identifies Morphogenesis-Associated Proteins during Cardiac Differentiation of Human iPS Cells. ACS OMEGA 2025; 10:344-357. [PMID: 39829588 PMCID: PMC11740111 DOI: 10.1021/acsomega.4c06371] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/09/2024] [Revised: 12/06/2024] [Accepted: 12/13/2024] [Indexed: 01/22/2025]
Abstract
Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have potential applications in regenerative medicine. The quality by design (QbD) approach enables the efficiency and quality assurance in the manufacturing of hiPSC-derived products. It requires a molecular understanding of hiPSC differentiation throughout the differentiation process; however, information on cardiac differentiation remains limited. Proteins associated with the early stages of cardiac differentiation would be useful in the cardiomyocyte quality assessment. Here, we performed quantitative proteomics of hiPSC intermediate cells in the early phase of cardiac differentiation to better understand their molecular characteristics. Proteomic profiles suggested that day 5-7 cells were in the morphogenetic stage of cardiac differentiation. Trophoblast glycoprotein (TPBG) was the most up-regulated protein in the morphogenetic stage; it was previously shown to be up-regulated during differentiation into neural stem cells. Proteomics of TPBG-knockdown cells revealed that TPBG is involved in cell proliferation and is related to the cardiomyocyte yield, suggesting that it could be used as a marker in QbD development. Our approach helps us understand the molecular basis of hiPSC differentiation and could be a powerful tool in QbD-based manufacturing.
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Affiliation(s)
- Takaya Urasawa
- Biopharmaceutical and Regenerative
Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
| | - Nana Kawasaki
- Biopharmaceutical and Regenerative
Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
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5
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Setsu S, Morimoto S, Nakamura S, Ozawa F, Utami KH, Nishiyama A, Suzuki N, Aoki M, Takeshita Y, Tomari Y, Okano H. Swift induction of human spinal lower motor neurons and robust ALS cell screening via single-cell imaging. Stem Cell Reports 2025; 20:102377. [PMID: 39706179 PMCID: PMC11784480 DOI: 10.1016/j.stemcr.2024.11.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Revised: 11/13/2024] [Accepted: 11/18/2024] [Indexed: 12/23/2024] Open
Abstract
This study introduces a novel method for rapidly and efficiently inducing human spinal lower motor neurons (LMNs) from induced pluripotent stem cells (iPSCs) to eventually elucidate the pathomechanisms of amyotrophic lateral sclerosis (ALS) and facilitate drug screening. Previous methods were limited by low induction efficiency, poor LMN purity, or labor-intensive induction and evaluation processes. Our protocol overcomes these challenges, achieving around 80% induction efficiency within just two weeks by combining a small molecule-based approach with transcription factor transduction. Moreover, to exclude non-LMN cells from the analysis, we utilized time-lapse microscopy and machine learning to analyze the morphology and viability of iPSC-derived LMNs on a single-cell basis, establishing an effective pathophysiological evaluation system. This rapid, efficient, and streamlined protocol, along with our single-cell-based evaluation method, enables large-scale analysis and drug screening using iPSC-derived motor neurons.
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Affiliation(s)
- Selena Setsu
- Keio University Regenerative Medicine Research Center, Kanagawa 210-0821, Japan; Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Satoru Morimoto
- Keio University Regenerative Medicine Research Center, Kanagawa 210-0821, Japan; Division of Neurodegenerative Disease Research, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Tokyo 173-0015, Japan.
| | - Shiho Nakamura
- Keio University Regenerative Medicine Research Center, Kanagawa 210-0821, Japan; Division of Neurodegenerative Disease Research, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Tokyo 173-0015, Japan
| | - Fumiko Ozawa
- Keio University Regenerative Medicine Research Center, Kanagawa 210-0821, Japan; Division of Neurodegenerative Disease Research, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Tokyo 173-0015, Japan
| | - Kagistia Hana Utami
- Keio University Regenerative Medicine Research Center, Kanagawa 210-0821, Japan; Division of Neurodegenerative Disease Research, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Tokyo 173-0015, Japan
| | - Ayumi Nishiyama
- Department of Neurology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
| | - Naoki Suzuki
- Department of Neurology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan; Department of Rehabilitation Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Masashi Aoki
- Department of Neurology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
| | - Yukio Takeshita
- Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Yamaguchi 753-8511, Japan; Department of Neurotherapeutics, Yamaguchi University Graduate School of Medicine, Yamaguchi 753-8511, Japan
| | - Yukihide Tomari
- Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Hideyuki Okano
- Keio University Regenerative Medicine Research Center, Kanagawa 210-0821, Japan; Division of Neurodegenerative Disease Research, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Tokyo 173-0015, Japan.
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Wen J, Li Y, Qin Y, Yan L, Zhang K, Li A, Wang Z, Yu F, Lai J, Yang W, Liu YU, Qin D, Su H. Lycorine protects motor neurons against TDP-43 proteinopathy-induced degeneration in cross-species models with amyotrophic lateral sclerosis. Pharmacol Res 2024; 210:107518. [PMID: 39603574 DOI: 10.1016/j.phrs.2024.107518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/19/2024] [Revised: 11/13/2024] [Accepted: 11/21/2024] [Indexed: 11/29/2024]
Abstract
Aggregation of TAR-DNA binding protein-43 (TDP-43) is a pathological feature present in nearly 97 % cases of amyotrophic lateral sclerosis (ALS), making it an attractive target for pathogenic studies and drug screening. Here, we have performed a high-throughput screening of 1500 compounds from a natural product library and identified that lycorine, a naturally occurring alkaloid, significantly decreases the level of TDP-43A315T in a cellular model. We further demonstrate that lycorine reduces the level of TDP-43A315T both through inhibiting its synthesis and by promoting its degradation by the ubiquitin-proteasome system (UPS). Importantly, treatment with lycorine significantly attenuates TDP-43 proteinopathy and improves functional recovery in TDP-43A315T-expressing Caenorhabditis elegans and mouse models. These findings suggest that lycorine is a promising lead compound that has therapeutic potential for ALS.
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Affiliation(s)
- Jing Wen
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Yunhao Li
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Yanzhu Qin
- Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510799, China
| | - Lingli Yan
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Ke Zhang
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Ang Li
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Ziying Wang
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Feng Yu
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao
| | - Jianheng Lai
- Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510799, China
| | - Wei Yang
- The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China; GuiZhou University Medical College, Guiyang 550025, China
| | - Yong U Liu
- Laboratory for Neuroimmunology in Health and Diseases, Center for Medical Research on Innovation and Translation, Institute of Clinical Medicine, the Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510180, China.
| | - Dajiang Qin
- Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510799, China.
| | - Huanxing Su
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao.
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Theme 5 Human Cell Biology and Pathology. Amyotroph Lateral Scler Frontotemporal Degener 2024; 25:158-184. [PMID: 39508672 DOI: 10.1080/21678421.2024.2403302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2024]
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Stillman JM, Kiniwa T, Schafer DP. Nucleic acid sensing in the central nervous system: Implications for neural circuit development, function, and degeneration. Immunol Rev 2024; 327:71-82. [PMID: 39503567 PMCID: PMC11653434 DOI: 10.1111/imr.13420] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2024]
Abstract
Nucleic acids are a critical trigger for the innate immune response to infection, wherein pathogen-derived RNA and DNA are sensed by nucleic acid sensing receptors. This subsequently drives the production of type I interferon and other inflammatory cytokines to combat infection. While the system is designed such that these receptors should specifically recognize pathogen-derived nucleic acids, it is now clear that self-derived RNA and DNA can also stimulate these receptors to cause aberrant inflammation and autoimmune disease. Intriguingly, similar pathways are now emerging in the central nervous system in neurons and glial cells. As in the periphery, these signaling pathways are active in neurons and glia to present the spread of pathogens in the CNS. They further appear to be active even under steady conditions to regulate neuronal development and function, and they can become activated aberrantly during disease to propagate neuroinflammation and neurodegeneration. Here, we review the emerging new roles for nucleic acid sensing mechanisms in the CNS and raise open questions that we are poised to explore in the future.
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Affiliation(s)
- Jacob M. Stillman
- Department of Neurobiology, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- University of Massachusetts Chan Morningside Graduate School of Biomedical Sciences, Neuroscience Program, Worcester, MA, USA
| | - Tsuyoshi Kiniwa
- Department of Neurobiology, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
| | - Dorothy P. Schafer
- Department of Neurobiology, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
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9
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Tsekrekou M, Giannakou M, Papanikolopoulou K, Skretas G. Protein aggregation and therapeutic strategies in SOD1- and TDP-43- linked ALS. Front Mol Biosci 2024; 11:1383453. [PMID: 38855322 PMCID: PMC11157337 DOI: 10.3389/fmolb.2024.1383453] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2024] [Accepted: 05/02/2024] [Indexed: 06/11/2024] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with severe socio-economic impact. A hallmark of ALS pathology is the presence of aberrant cytoplasmic inclusions composed of misfolded and aggregated proteins, including both wild-type and mutant forms. This review highlights the critical role of misfolded protein species in ALS pathogenesis, particularly focusing on Cu/Zn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43), and emphasizes the urgent need for innovative therapeutic strategies targeting these misfolded proteins directly. Despite significant advancements in understanding ALS mechanisms, the disease remains incurable, with current treatments offering limited clinical benefits. Through a comprehensive analysis, the review focuses on the direct modulation of the misfolded proteins and presents recent discoveries in small molecules and peptides that inhibit SOD1 and TDP-43 aggregation, underscoring their potential as effective treatments to modify disease progression and improve clinical outcomes.
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Affiliation(s)
- Maria Tsekrekou
- Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece
| | - Maria Giannakou
- Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece
- Department of Biology, National and Kapodistrian University of Athens, Athens, Greece
| | - Katerina Papanikolopoulou
- Institute for Fundamental Biomedical Research, Biomedical Sciences Research Centre “Alexander Fleming”, Vari, Greece
- ResQ Biotech, Patras Science Park, Rio, Greece
| | - Georgios Skretas
- Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece
- ResQ Biotech, Patras Science Park, Rio, Greece
- Institute for Bio-innovation, Biomedical Sciences Research Centre “Alexander Fleming”, Vari, Greece
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10
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Cerneckis J, Cai H, Shi Y. Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications. Signal Transduct Target Ther 2024; 9:112. [PMID: 38670977 PMCID: PMC11053163 DOI: 10.1038/s41392-024-01809-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 03/09/2024] [Accepted: 03/17/2024] [Indexed: 04/28/2024] Open
Abstract
The induced pluripotent stem cell (iPSC) technology has transformed in vitro research and holds great promise to advance regenerative medicine. iPSCs have the capacity for an almost unlimited expansion, are amenable to genetic engineering, and can be differentiated into most somatic cell types. iPSCs have been widely applied to model human development and diseases, perform drug screening, and develop cell therapies. In this review, we outline key developments in the iPSC field and highlight the immense versatility of the iPSC technology for in vitro modeling and therapeutic applications. We begin by discussing the pivotal discoveries that revealed the potential of a somatic cell nucleus for reprogramming and led to successful generation of iPSCs. We consider the molecular mechanisms and dynamics of somatic cell reprogramming as well as the numerous methods available to induce pluripotency. Subsequently, we discuss various iPSC-based cellular models, from mono-cultures of a single cell type to complex three-dimensional organoids, and how these models can be applied to elucidate the mechanisms of human development and diseases. We use examples of neurological disorders, coronavirus disease 2019 (COVID-19), and cancer to highlight the diversity of disease-specific phenotypes that can be modeled using iPSC-derived cells. We also consider how iPSC-derived cellular models can be used in high-throughput drug screening and drug toxicity studies. Finally, we discuss the process of developing autologous and allogeneic iPSC-based cell therapies and their potential to alleviate human diseases.
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Affiliation(s)
- Jonas Cerneckis
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
- Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
| | - Hongxia Cai
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
| | - Yanhong Shi
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
- Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
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11
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Garcia EL, Steiner RE, Raimer AC, Herring LE, Matera AG, Spring AM. Dysregulation of innate immune signaling in animal models of spinal muscular atrophy. BMC Biol 2024; 22:94. [PMID: 38664795 PMCID: PMC11044505 DOI: 10.1186/s12915-024-01888-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2023] [Accepted: 04/16/2024] [Indexed: 04/28/2024] Open
Abstract
BACKGROUND Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the survival motor neuron (SMN) protein. SMA presents across a broad spectrum of disease severity. Unfortunately, genetic models of intermediate SMA have been difficult to generate in vertebrates and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes. RESULTS Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the immune deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, the knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of a ubiquitylation complex that includes Traf6, Bendless, and Diap2 and plays a pivotal role in several signaling networks. CONCLUSIONS In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.
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Affiliation(s)
- Eric L Garcia
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
- Department of Biology, University of Kentucky, Lexington, KY, USA
| | - Rebecca E Steiner
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
- RNA Discovery and Lineberger Comprehensive Cancer Centers, University of North Carolina at Chapel Hill, Chapel Hill, 27599, USA
- Present Address: Lake, Erie College of Osteopathic Medicine, Bradenton, FL, USA
| | - Amanda C Raimer
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, USA
- Present Address, Radford University, Radford, VA, USA
| | - Laura E Herring
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, USA
| | - A Gregory Matera
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, USA.
- Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, USA.
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, 27599, USA.
- RNA Discovery and Lineberger Comprehensive Cancer Centers, University of North Carolina at Chapel Hill, Chapel Hill, 27599, USA.
| | - Ashlyn M Spring
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
- Department of Biology, University of North Carolina at Greensboro, Greensboro, NC, 27402, USA.
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12
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De Cock L, Bercier V, Van Den Bosch L. New developments in pre-clinical models of ALS to guide translation. INTERNATIONAL REVIEW OF NEUROBIOLOGY 2024; 176:477-524. [PMID: 38802181 DOI: 10.1016/bs.irn.2024.04.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder in which selective death of motor neurons leads to muscle weakness and paralysis. Most research has focused on understanding and treating monogenic familial forms, most frequently caused by mutations in SOD1, FUS, TARDBP and C9orf72, although ALS is mostly sporadic and without a clear genetic cause. Rodent models have been developed to study monogenic ALS, but despite numerous pre-clinical studies and clinical trials, few disease-modifying therapies are available. ALS is a heterogeneous disease with complex underlying mechanisms where several genes and molecular pathways appear to play a role. One reason for the high failure rate of clinical translation from the current models could be oversimplification in pre-clinical studies. Here, we review advances in pre-clinical models to better capture the heterogeneous nature of ALS and discuss the value of novel model systems to guide translation and aid in the development of precision medicine.
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Affiliation(s)
- Lenja De Cock
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain-University of Leuven, Leuven, Belgium; Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
| | - Valérie Bercier
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain-University of Leuven, Leuven, Belgium; Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
| | - Ludo Van Den Bosch
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain-University of Leuven, Leuven, Belgium; Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium.
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13
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Chen Y, Li M, Wu Y. The occurrence and development of induced pluripotent stem cells. Front Genet 2024; 15:1389558. [PMID: 38699229 PMCID: PMC11063328 DOI: 10.3389/fgene.2024.1389558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Accepted: 04/08/2024] [Indexed: 05/05/2024] Open
Abstract
The ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc (OSKM), known as "Yamanaka factors," can reprogram or stimulate the production of induced pluripotent stem cells (iPSCs). Although OSKM is still the gold standard, there are multiple ways to reprogram cells into iPSCs. In recent years, significant progress has been made in improving the efficiency of this technology. Ten years after the first report was published, human pluripotent stem cells have gradually been applied in clinical settings, including disease modeling, cell therapy, new drug development, and cell derivation. Here, we provide a review of the discovery of iPSCs and their applications in disease and development.
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Affiliation(s)
| | - Meng Li
- Department of Cardiology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Yanqing Wu
- Department of Cardiology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China
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14
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Lépine S, Nauleau-Javaudin A, Deneault E, Chen CXQ, Abdian N, Franco-Flores AK, Haghi G, Castellanos-Montiel MJ, Maussion G, Chaineau M, Durcan TM. Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons. iScience 2024; 27:109166. [PMID: 38433895 PMCID: PMC10905001 DOI: 10.1016/j.isci.2024.109166] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 11/21/2023] [Accepted: 02/05/2024] [Indexed: 03/05/2024] Open
Abstract
Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in TARDBP (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9, we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease.
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Affiliation(s)
- Sarah Lépine
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
- Faculty of Medicine and Health Sciences, McGill University, Montreal, QC H3G 2M1, Canada
| | - Angela Nauleau-Javaudin
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
- Faculty of Medicine, Université de Montréal, Montreal, QC H3C 3J7, Canada
| | - Eric Deneault
- Centre for Oncology, Radiopharmaceuticals and Research; Biologic and Radiopharmaceutical Drugs Directorate, Health Products and Food Branch, Health Canada, Ottawa, ON K1A 0K9, Canada
| | - Carol X.-Q. Chen
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - Narges Abdian
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - Anna Krystina Franco-Flores
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - Ghazal Haghi
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - María José Castellanos-Montiel
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - Gilles Maussion
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - Mathilde Chaineau
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
| | - Thomas Martin Durcan
- Early Drug Discovery Unit (EDDU), The Neuro-Montreal Neurological Institute and Hospital, Department of Neurology and Neurosurgery, McGill University, Montreal, QC H3A 1A1, Canada
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15
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Suzuki H, Egawa N, Imamura K, Kondo T, Enami T, Tsukita K, Suga M, Yada Y, Shibukawa R, Takahashi R, Inoue H. Mutant α-synuclein causes death of human cortical neurons via ERK1/2 and JNK activation. Mol Brain 2024; 17:14. [PMID: 38444039 PMCID: PMC10916047 DOI: 10.1186/s13041-024-01086-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2023] [Accepted: 02/26/2024] [Indexed: 03/07/2024] Open
Abstract
Synucleinopathies refer to a group of disorders characterized by SNCA/α-synuclein (α-Syn)-containing cytoplasmic inclusions and neuronal cell loss in the nervous system including the cortex, a common feature being cognitive impairment. Still, the molecular pathogenesis of cognitive decline remains poorly understood, hampering the development of effective treatments. Here, we generated induced pluripotent stem cells (iPSCs) derived from familial Parkinson's disease (PD) patients carrying SNCA A53T mutation, differentiating them into cortical neurons by a direct conversion method. Patient iPSCs-derived cortical neurons harboring mutant α-Syn exhibited increased α-Syn-positive aggregates, shorter neurites, and time-dependent vulnerability. Furthermore, RNA-sequencing analysis, followed by biochemical validation, identified the activation of the ERK1/2 and JNK cascades in cortical neurons with SNCA A53T mutation. This result was consistent with a reverted phenotype of neuronal death in cortical neurons when treated with ERK1/2 and JNK inhibitors, respectively. Our findings emphasize the role of ERK1/2 and JNK cascades in the vulnerability of cortical neurons in synucleinopathies, and they could pave the way toward therapeutic advancements for synucleinopathies.
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Affiliation(s)
- Hidefumi Suzuki
- Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Naohiro Egawa
- Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Keiko Imamura
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Medical-Risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Takayuki Kondo
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Medical-Risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Takako Enami
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Medical-Risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan
| | - Kayoko Tsukita
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Mika Suga
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Yuichiro Yada
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Ran Shibukawa
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan
| | - Ryosuke Takahashi
- Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan.
| | - Haruhisa Inoue
- iPSC-Based Drug Discovery and Development Team, RIKEN BioResource Research Center (BRC), Kyoto, Japan.
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
- Medical-Risk Avoidance Based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan.
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16
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Miwata T, Suga H, Mitsumoto K, Zhang J, Hamada Y, Sakakibara M, Soen M, Ozaki H, Asano T, Miyata T, Kawaguchi Y, Yasuda Y, Kobayashi T, Sugiyama M, Onoue T, Hagiwara D, Iwama S, Oyadomari S, Arima H. Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells. Peptides 2024; 173:171151. [PMID: 38215943 DOI: 10.1016/j.peptides.2024.171151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 12/17/2023] [Accepted: 01/08/2024] [Indexed: 01/14/2024]
Abstract
Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.
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Affiliation(s)
- Tsutomu Miwata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hidetaka Suga
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan.
| | - Kazuki Mitsumoto
- Department of Endocrinology and Diabetes, Gifu Prefectural Tajimi Hospital, Tajimi, Japan
| | - Jun Zhang
- Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Yoshimasa Hamada
- Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Mayu Sakakibara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mika Soen
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Hajime Ozaki
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tomoyoshi Asano
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Takashi Miyata
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yohei Kawaguchi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yoshinori Yasuda
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Tomoko Kobayashi
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mariko Sugiyama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Takeshi Onoue
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Daisuke Hagiwara
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Shintaro Iwama
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Seiichi Oyadomari
- Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Hiroshi Arima
- Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan
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17
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Zhang T, Qian C, Song M, Tang Y, Zhou Y, Dong G, Shen Q, Chen W, Wang A, Shen S, Zhao Y, Lu Y. Application Prospect of Induced Pluripotent Stem Cells in Organoids and Cell Therapy. Int J Mol Sci 2024; 25:2680. [PMID: 38473926 DOI: 10.3390/ijms25052680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Revised: 02/13/2024] [Accepted: 02/23/2024] [Indexed: 03/14/2024] Open
Abstract
Since its inception, induced pluripotent stem cell (iPSC) technology has been hailed as a powerful tool for comprehending disease etiology and advancing drug screening across various domains. While earlier iPSC-based disease modeling and drug assessment primarily operated at the cellular level, recent years have witnessed a significant shift towards organoid-based investigations. Organoids derived from iPSCs offer distinct advantages, particularly in enabling the observation of disease progression and drug metabolism in an in vivo-like environment, surpassing the capabilities of iPSC-derived cells. Furthermore, iPSC-based cell therapy has emerged as a focal point of clinical interest. In this review, we provide an extensive overview of non-integrative reprogramming methods that have evolved since the inception of iPSC technology. We also deliver a comprehensive examination of iPSC-derived organoids, spanning the realms of the nervous system, cardiovascular system, and oncology, as well as systematically elucidate recent advancements in iPSC-related cell therapies.
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Affiliation(s)
- Teng Zhang
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Cheng Qian
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Mengyao Song
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Yu Tang
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Yueke Zhou
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Guanglu Dong
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Department of Biochemistry and Molecular Biology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Qiuhong Shen
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Department of Biochemistry and Molecular Biology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Wenxing Chen
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Aiyun Wang
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Sanbing Shen
- Regenerative Medicine Institute, School of Medicine, University of Galway, H91 W2TY Galway, Ireland
| | - Yang Zhao
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Department of Biochemistry and Molecular Biology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
| | - Yin Lu
- Jiangsu Joint International Research Laboratory of Chinese Medicine and Regenerative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
- Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China
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18
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Beghini DG, Kasai-Brunswick TH, Henriques-Pons A. Induced Pluripotent Stem Cells in Drug Discovery and Neurodegenerative Disease Modelling. Int J Mol Sci 2024; 25:2392. [PMID: 38397069 PMCID: PMC10889263 DOI: 10.3390/ijms25042392] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2023] [Revised: 12/28/2023] [Accepted: 12/29/2023] [Indexed: 02/25/2024] Open
Abstract
Induced pluripotent stem cells (iPSCs) are derived from reprogrammed adult somatic cells. These adult cells are manipulated in vitro to express genes and factors essential for acquiring and maintaining embryonic stem cell (ESC) properties. This technology is widely applied in many fields, and much attention has been given to developing iPSC-based disease models to validate drug discovery platforms and study the pathophysiological molecular processes underlying disease onset. Especially in neurological diseases, there is a great need for iPSC-based technological research, as these cells can be obtained from each patient and carry the individual's bulk of genetic mutations and unique properties. Moreover, iPSCs can differentiate into multiple cell types. These are essential characteristics, since the study of neurological diseases is affected by the limited access to injury sites, the need for in vitro models composed of various cell types, the complexity of reproducing the brain's anatomy, the challenges of postmortem cell culture, and ethical issues. Neurodegenerative diseases strongly impact global health due to their high incidence, symptom severity, and lack of effective therapies. Recently, analyses using disease specific, iPSC-based models confirmed the efficacy of these models for testing multiple drugs. This review summarizes the advances in iPSC technology used in disease modelling and drug testing, with a primary focus on neurodegenerative diseases, including Parkinson's and Alzheimer's diseases.
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Affiliation(s)
- Daniela Gois Beghini
- Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro 21040-900, RJ, Brazil;
| | - Tais Hanae Kasai-Brunswick
- Centro Nacional de Biologia Estrutural e Bioimagem, CENABIO, Universidade Federal do Rio de Janeiro, Seropédica 23890-000, RJ, Brazil;
- Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa, INCT-REGENERA, Universidade Federal do Rio de Janeiro, Seropédica 23890-000, RJ, Brazil
| | - Andrea Henriques-Pons
- Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro 21040-900, RJ, Brazil;
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Xin J, Huang S, Wen J, Li Y, Li A, Satyanarayanan SK, Yao X, Su H. Drug Screening and Validation Targeting TDP-43 Proteinopathy for Amyotrophic Lateral Sclerosis. Aging Dis 2024; 16:693-713. [PMID: 38739934 PMCID: PMC11964425 DOI: 10.14336/ad.2024.0440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2024] [Accepted: 05/05/2024] [Indexed: 05/16/2024] Open
Abstract
Amyotrophic lateral sclerosis (ALS) stands as a rare, yet severely debilitating disorder marked by the deterioration of motor neurons (MNs) within the brain and spinal cord, which is accompanied by degenerated corticobulbar/corticospinal tracts and denervation in skeletal muscles. Despite ongoing research efforts, ALS remains incurable, attributed to its intricate pathogenic mechanisms. A notable feature in the pathology of ALS is the prevalence of TAR DNA-binding protein 43 (TDP-43) proteinopathy, detected in approximately 97% of ALS cases, underscoring its significance in the disease's progression. As a result, strategies targeting the aberrant TDP-43 protein have garnered attention as a potential avenue for ALS therapy. This review delves into the existing drug screening systems aimed at TDP-43 proteinopathy and the models employed for drug efficacy validation. It also explores the hurdles encountered in the quest to develop potent medications against TDP-43 proteinopathy, offering insights into the intricacies of drug discovery and development for ALS. Through this comprehensive analysis, the review sheds light on the critical aspects of identifying and advancing therapeutic solutions for ALS.
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Affiliation(s)
- Jiaqi Xin
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
| | - Sen Huang
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University; Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases; National Key Clinical Department and Key Discipline of Neurology, Guangzhou, China.
| | - Jing Wen
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
| | - Yunhao Li
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
| | - Ang Li
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
| | - Senthil Kumaran Satyanarayanan
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong Science Park, Hong Kong, China.
| | - Xiaoli Yao
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University; Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases; National Key Clinical Department and Key Discipline of Neurology, Guangzhou, China.
| | - Huanxing Su
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
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20
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Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Front Cell Neurosci 2024; 17:1327361. [PMID: 38314348 PMCID: PMC10834640 DOI: 10.3389/fncel.2023.1327361] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2023] [Accepted: 12/15/2023] [Indexed: 02/06/2024] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron (MN) disease with severely limited treatment options. Identification of effective treatments has been limited in part by the lack of predictive animal models for complex human disorders. Here, we utilized pharmacologic ER stressors to exacerbate underlying sensitivities conferred by ALS patient genetics in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). In doing so, we found that thapsigargin and tunicamycin exposure recapitulated ALS-associated degeneration, and that we could rescue this degeneration via MAP4K4 inhibition (MAP4K4i). We subsequently identified mechanisms underlying MAP4K4i-mediated protection by performing phosphoproteomics on iPSC-derived MNs treated with ER stressors ±MAP4K4i. Through these analyses, we found JNK, PKC, and BRAF to be differentially modulated in MAP4K4i-protected MNs, and that inhibitors to these proteins could also rescue MN toxicity. Collectively, this study highlights the value of utilizing ER stressors in ALS patient MNs to identify novel druggable targets.
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Affiliation(s)
- Michelle E. Watts
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, United States
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA, United States
| | - Richard M. Giadone
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, United States
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA, United States
| | - Alban Ordureau
- Department of Cell Biology, Harvard Medical School, Boston, MA, United States
| | - Kristina M. Holton
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, United States
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA, United States
| | - J. Wade Harper
- Department of Cell Biology, Harvard Medical School, Boston, MA, United States
| | - Lee L. Rubin
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, United States
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA, United States
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Paris A, Lakatos A. Cell and gene therapy for amyotrophic lateral sclerosis. HANDBOOK OF CLINICAL NEUROLOGY 2024; 205:217-241. [PMID: 39341656 DOI: 10.1016/b978-0-323-90120-8.00017-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/01/2024]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disorder with rapidly progressive skeletal muscle weakness, which can also cause a variable cognitive deficit. Genetic causes are only identified in approximately 10% of all cases, with complex genotype-phenotype associations, making it challenging to identify treatment targets. What further hampers therapeutic development is a broad heterogeneity in mechanisms, possible targets, and disturbances across various cell types, aside from the cortical and spinal motor neurons that lie at the heart of the pathology of ALS. Over the last decade, significant progress in biotechnologic techniques, cell and ribonucleic acid (RNA) engineering, animal models, and patient-specific human stem cell and organoid models have accelerated both mechanistic and therapeutic discoveries. The growing number of clinical trials mirrors this. This chapter reviews the current state of human preclinical models supporting trial strategies as well as recent clinical cell and gene therapy approaches.
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Affiliation(s)
- Alvar Paris
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom; Department of Neurology, Cambridge University Hospitals NHS Trust, Addenbrooke's Hospital, Cambridge, United Kingdom
| | - András Lakatos
- Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom; Department of Neurology, Cambridge University Hospitals NHS Trust, Addenbrooke's Hospital, Cambridge, United Kingdom.
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22
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Kusama-Eguchi K, Tokui Y, Minoura A, Yanai Y, Hirose D, Furukawa M, Kosuge Y, Miura M, Ohkoshi E, Makino M, Minagawa K, Matsuzaki K, Ogawa Y, Watanabe K, Ohsaki A. 2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs. J Nat Med 2024; 78:146-159. [PMID: 37804412 DOI: 10.1007/s11418-023-01751-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2023] [Accepted: 09/08/2023] [Indexed: 10/09/2023]
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.
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Affiliation(s)
- Kuniko Kusama-Eguchi
- Department of Chemistry, College of Humanities and Science, Ninon University, Setagaya-Ku, Tokyo, 156-8550, Japan.
- Laboratory of Biochemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan.
- Laboratory of Medical Microbiology, School of Pharmacy, Nihon University. Funabashi, Chiba, 274-8555, Japan.
| | - Yuki Tokui
- Department of Chemistry, College of Humanities and Science, Ninon University, Setagaya-Ku, Tokyo, 156-8550, Japan
| | - Ai Minoura
- Laboratory of Biochemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Yuta Yanai
- Department of Chemistry, College of Humanities and Science, Ninon University, Setagaya-Ku, Tokyo, 156-8550, Japan
- Laboratory of Biochemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Dai Hirose
- Laboratory of Medical Microbiology, School of Pharmacy, Nihon University. Funabashi, Chiba, 274-8555, Japan
| | - Megumi Furukawa
- Laboratory of Pharmacognosy, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Yasuhiro Kosuge
- Laboratory of Pharmacology, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Motofumi Miura
- Laboratory of Molecular Chemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Emika Ohkoshi
- Department of Natural Products Chemistry, Faculty of Pharmaceutical Sciences, Aomori University, Aomori, Aomori, 030-0943, Japan
| | - Mitsuko Makino
- Laboratory of Pharmacognosy, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Kimino Minagawa
- Laboratory of Biochemistry, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
- Division of Genomic Epidemiology and Clinical Trials, Clinical Trials Research Center, Nihon University School of Medicine, Tokyo, Japan
| | - Keiichi Matsuzaki
- Laboratory of Pharmacognosy, School of Pharmacy, Nihon University, Funabashi, Chiba, 274-8555, Japan
| | - Yoshio Ogawa
- Laboratory of Medical Microbiology, School of Pharmacy, Nihon University. Funabashi, Chiba, 274-8555, Japan
| | - Kazuko Watanabe
- Laboratory of Medical Microbiology, School of Pharmacy, Nihon University. Funabashi, Chiba, 274-8555, Japan
| | - Ayumi Ohsaki
- Department of Chemistry, College of Humanities and Science, Ninon University, Setagaya-Ku, Tokyo, 156-8550, Japan.
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23
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Garcia EL, Steiner RE, Raimer AC, Herring LE, Matera AG, Spring AM. Dysregulation of innate immune signaling in animal models of Spinal Muscular Atrophy. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.14.571739. [PMID: 38168196 PMCID: PMC10760185 DOI: 10.1101/2023.12.14.571739] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Background Spinal Muscular Atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the Survival Motor Neuron (SMN) protein. SMA presents across broad spectrum of disease severity. Unfortunately, vertebrate models of intermediate SMA have been difficult to generate and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes. Results Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the Immune Deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of an ubiquitylation complex that includes Traf6, Bendless and Diap2, and plays a pivotal role in several signaling networks. Conclusions In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.
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Affiliation(s)
- Eric L. Garcia
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill NC, USA
- Department of Biology, University of Kentucky, Lexington KY, USA
| | - Rebecca E. Steiner
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill NC, USA
- Department of Biology, University of North Carolina at Chapel Hill
| | - Amanda C. Raimer
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill NC, USA
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill
| | - Laura E. Herring
- Department of Pharmacology, University of North Carolina at Chapel Hill
| | - A. Gregory Matera
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill NC, USA
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill
- Department of Biology, University of North Carolina at Chapel Hill
- Department of Genetics, University of North Carolina at Chapel Hill
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill
| | - Ashlyn M. Spring
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill NC, USA
- Department of Biology, University of North Carolina at Greensboro, Greensboro NC, USA
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24
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Elmansy MF, Reidl CT, Rahaman M, Özdinler PH, Silverman RB. Small molecules targeting different cellular pathologies for the treatment of amyotrophic lateral sclerosis. Med Res Rev 2023; 43:2260-2302. [PMID: 37243319 PMCID: PMC10592673 DOI: 10.1002/med.21974] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Revised: 02/28/2023] [Accepted: 04/30/2023] [Indexed: 05/28/2023]
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease in which the motor neuron circuitry displays progressive degeneration, affecting mostly the motor neurons in the brain and in the spinal cord. There are no effective cures, albeit three drugs, riluzole, edaravone, and AMX0035 (a combination of sodium phenylbutyrate and taurursodiol), have been approved by the Food and Drug Administration, with limited improvement in patients. There is an urgent need to build better and more effective treatment strategies for ALS. Since the disease is very heterogenous, numerous approaches have been explored, such as targeting genetic mutations, decreasing oxidative stress and excitotoxicity, enhancing mitochondrial function and protein degradation mechanisms, and inhibiting neuroinflammation. In addition, various chemical libraries or previously identified drugs have been screened for potential repurposing in the treatment of ALS. Here, we review previous drug discovery efforts targeting a variety of cellular pathologies that occur from genetic mutations that cause ALS, such as mutations in SOD1, C9orf72, FUS, and TARDP-43 genes. These mutations result in protein aggregation, which causes neuronal degeneration. Compounds used to target cellular pathologies that stem from these mutations are discussed and comparisons among different preclinical models are presented. Because the drug discovery landscape for ALS and other motor neuron diseases is changing rapidly, we also offer recommendations for a novel, more effective, direction in ALS drug discovery that could accelerate translation of effective compounds from animals to patients.
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Affiliation(s)
- Mohamed F. Elmansy
- Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois, USA
- Department of Organometallic and Organometalloid Chemistry, National Research Centre, Cairo, Egypt
| | - Cory T. Reidl
- Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois, USA
| | - Mizzanoor Rahaman
- Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois, USA
| | - P. Hande Özdinler
- Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
| | - Richard B. Silverman
- Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois, USA
- Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
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25
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Shinde P, Shinde A, Kar S, Illath K, Nagai M, Tseng FG, Santra TS. Ultrathin SU-8 membrane for highly efficient tunable cell patterning and massively parallel large biomolecular delivery. LAB ON A CHIP 2023; 23:4636-4651. [PMID: 37655799 DOI: 10.1039/d3lc00244f] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/02/2023]
Abstract
Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 μm. It features an array of micro-holes ranging from 40 μm to 80 μm, spaced apart by 50 μm to 150 μm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 μm outer diameter, 3 μm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and β-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.
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Affiliation(s)
- Pallavi Shinde
- Department of Engineering Design, Indian Institute of Technology Madras, Chennai, India.
| | - Ashwini Shinde
- Department of Engineering Design, Indian Institute of Technology Madras, Chennai, India.
| | - Srabani Kar
- Department of Physics, Indian Institute of Science Education and Research, Tirupati, India
| | - Kavitha Illath
- Department of Engineering Design, Indian Institute of Technology Madras, Chennai, India.
| | - Moeto Nagai
- Department of Mechanical Engineering, Toyohashi University of Technology, Japan
| | - Fan-Gang Tseng
- Department of Engineering and System Science, National Tsing Hua University, Taiwan
| | - Tuhin Subhra Santra
- Department of Engineering Design, Indian Institute of Technology Madras, Chennai, India.
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26
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Fang M, Deibler SK, Nana AL, Vatsavayai SC, Banday S, Zhou Y, Almeida S, Weiss A, Brown RH, Seeley WW, Gao FB, Green MR. Loss of TDP-43 function contributes to genomic instability in amyotrophic lateral sclerosis. Front Neurosci 2023; 17:1251228. [PMID: 37849894 PMCID: PMC10577185 DOI: 10.3389/fnins.2023.1251228] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2023] [Accepted: 09/08/2023] [Indexed: 10/19/2023] Open
Abstract
A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the cytoplasmic mislocalization and aggregation of the DNA/RNA-binding protein TDP-43, but how loss of nuclear TDP-43 function contributes to ALS and FTD pathogenesis remains largely unknown. Here, using large-scale RNAi screening, we identify TARDBP, which encodes TDP-43, as a gene whose loss-of-function results in elevated DNA mutation rate and genomic instability. Consistent with this finding, we observe increased DNA damage in induced pluripotent stem cells (iPSCs) and iPSC-derived post-mitotic neurons generated from ALS patients harboring TARDBP mutations. We find that the increase in DNA damage in ALS iPSC-derived neurons is due to defects in two major pathways for DNA double-strand break repair: non-homologous end joining and homologous recombination. Cells with defects in DNA repair are sensitive to DNA damaging agents and, accordingly, we find that ALS iPSC-derived neurons show a marked reduction in survival following treatment with a DNA damaging agent. Importantly, we find that increased DNA damage is also observed in neurons with nuclear TDP-43 depletion from ALS/FTD patient brain tissues. Collectively, our results demonstrate that ALS neurons with loss of nuclear TDP-43 function have elevated levels of DNA damage and contribute to the idea that genomic instability is a defining pathological feature of ALS/FTD patients with TDP-43 pathology.
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Affiliation(s)
- Minggang Fang
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Sara K. Deibler
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Alissa L. Nana
- Department of Neurology, Memory and Aging Center, University of California, San Francisco, San Francisco, CA, United States
| | - Sarat C. Vatsavayai
- Department of Neurology, Memory and Aging Center, University of California, San Francisco, San Francisco, CA, United States
| | - Shahid Banday
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - You Zhou
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Sandra Almeida
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Alexandra Weiss
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Robert H. Brown
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - William W. Seeley
- Department of Neurology, Memory and Aging Center, University of California, San Francisco, San Francisco, CA, United States
| | - Fen-Biao Gao
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA, United States
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, United States
| | - Michael R. Green
- Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, United States
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27
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Namatame I, Ishii K, Shin T, Shimojo D, Yamagishi Y, Asano H, Kishimoto Y, Fuse H, Nishi Y, Sakurai H, Nakahata T, Sasaki-Iwaoka H. Screening Station, a novel laboratory automation system for physiologically relevant cell-based assays. SLAS Technol 2023; 28:351-360. [PMID: 37121549 DOI: 10.1016/j.slast.2023.04.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2022] [Revised: 03/11/2023] [Accepted: 04/24/2023] [Indexed: 05/02/2023]
Abstract
Due to their physiological relevance, cell-based assays using human-induced pluripotent stem cell (iPSC)-derived cells are a promising in vitro pharmacological evaluation system for drug candidates. However, cell-based assays involve complex processes such as long-term culture, real-time and continuous observation of living cells, and detection of many cellular events. Automating multi-sample processing through these assays will enhance reproducibility by limiting human error and reduce researchers' valuable time spent conducting these experiments. Furthermore, this integration enables continuous tracking of morphological changes, which is not possible with the use of stand-alone devices. This report describes a new laboratory automation system called the Screening Station, which uses novel automation control and scheduling software called Green Button Go to integrate various devices. To integrate the above-mentioned processes, we established three workflows in Green Button Go: 1) For long-term cell culture, culture plates and medium containers are transported from the automatic CO2 incubator and cool incubator, respectively, and the cell culture medium in the microplates is exchanged daily using the Biomek i7 workstation; 2) For time-lapse live-cell imaging, culture plates are automatically transferred between the CQ1 confocal quantitative image cytometer and the SCALE48W automatic CO2 incubator; 3) For immunofluorescence imaging assays, in addition to the above-mentioned devices, the 405LS microplate washer allows for formalin-fixation and immunostaining of cells. By scheduling various combinations of the three workflows, we successfully automated the culture and medium exchange processes for iPSCs derived from patients with facioscapulohumeral muscular dystrophy, confirmation of their differentiation status by live-cell imaging, and confirmation of the presence of differentiation markers by immunostaining. In addition, deep learning analysis enabled us to quantify the degree of iPSC differentiation from live-cell imaging data. Further, the results of the fully automated experiments could be accessed via the intranet, enabling experiments and analysis to be conducted remotely once the necessary reagents and labware were prepared. We expect that the ability to perform clinically and physiologically relevant cell-based assays from remote locations using the Screening Station will facilitate global research collaboration and accelerate the discovery of new drug candidates.
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Affiliation(s)
- Ichiji Namatame
- Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan.
| | - Kana Ishii
- Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
| | - Takashi Shin
- Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
| | - Daisuke Shimojo
- Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
| | - Yukiko Yamagishi
- Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan; Center for iPS Cell Research and Application (CiRA), Kyoto University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Hidemitsu Asano
- Rorze Lifescience Inc., 430-1, Kamiyokoba, Tsukuba-shi, Ibaraki, 305-0854, Japan
| | - Yuuki Kishimoto
- Yokogawa Electric Co., 2-9-32, Naka-machi, Musashino-shi, Tokyo, 180-8750, Japan
| | - Hiromitsu Fuse
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Yohei Nishi
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Hidetoshi Sakurai
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Tatsutoshi Nakahata
- Center for iPS Cell Research and Application (CiRA), Kyoto University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
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28
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Soumya BS, Shreenidhi VP, Agarwal A, Gandhirajan RK, Dharmarajan A, Warrier S. Unwinding the role of Wnt signaling cascade and molecular triggers of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Cell Signal 2023; 110:110807. [PMID: 37463628 DOI: 10.1016/j.cellsig.2023.110807] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2023] [Revised: 07/07/2023] [Accepted: 07/13/2023] [Indexed: 07/20/2023]
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative condition, triggered by various factors causing the degeneration of upper and lower motor neurons, resulting in progressive muscle wasting, paralysis, and death. Multiple in vivo and in vitro models have been established to unravel the molecular events leading to the deterioration of motor neurons in ALS. The canonical and non-canonical Wnt signaling pathway has been implicated to play a crucial role in the progression of neurodegenerative disorders. This review discusses the role of Wnt signaling in the reported causes of ALS such as oxidative stress, mitochondrial dysfunction, autophagy, and apoptosis. Mutations in ALS-associated genes such as SOD1, C9orf72, TDP43, FUS, and OPTN cause an imbalance in neuronal integrity and homeostasis leading to motor neuron demise. Wnt signaling is also observed to play a crucial role in the muscle sparing of oculomotor neurons. The non-canonical Wnt/Ca2+ pathway which regulates intrinsic electrophysiological properties and mobilizes calcium ions to maintain neuronal integrity has been found to be altered in the stem cell-derived ALS model. Thus, the interplay of dysregulated canonical and non-canonical Wnt pathways in multiple motor neuron disease models has shown that Wnt contributes to disease progression indicating it to be utilized as a potential target for ALS.
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Affiliation(s)
- B S Soumya
- Division of Cancer Stem Cells and Cardiovascular Regeneration, Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education (MAHE), Bangalore 560065, India
| | - V P Shreenidhi
- Division of Cancer Stem Cells and Cardiovascular Regeneration, Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education (MAHE), Bangalore 560065, India
| | - Apoorvaa Agarwal
- Division of Cancer Stem Cells and Cardiovascular Regeneration, Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education (MAHE), Bangalore 560065, India
| | - Rajesh Kumar Gandhirajan
- Department of Human Genetics, Faculty of Biomedical Sciences and Technology, Sri Ramachandra Institute of Higher Education and Research, Chennai 600116, India
| | - Arun Dharmarajan
- Department of Biomedical Sciences, Faculty of Biomedical Sciences and Technology, Sri Ramachandra Institute of Higher Education and Research, Chennai 600116, India; School of Human Sciences, Faculty of Life and Physical Sciences, The University of Western Australia, Perth, Australia; Curtin Medical School, Curtin University, Perth, Western Australia, Australia
| | - Sudha Warrier
- Division of Cancer Stem Cells and Cardiovascular Regeneration, Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education (MAHE), Bangalore 560065, India; Cuor Stem Cellutions Pvt Ltd, Manipal Institute of Regenerative Medicine, Manipal Academy of Higher Education (MAHE), Bangalore 560065, India; Department of Biotechnology, Faculty of Biomedical Sciences and Technology, Sri Ramachandra Institute of Higher Education and Research, Chennai 600116, India.
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29
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Beckers J, Tharkeshwar AK, Fumagalli L, Contardo M, Van Schoor E, Fazal R, Thal DR, Chandran S, Mancuso R, Van Den Bosch L, Van Damme P. A toxic gain-of-function mechanism in C9orf72 ALS impairs the autophagy-lysosome pathway in neurons. Acta Neuropathol Commun 2023; 11:151. [PMID: 37723585 PMCID: PMC10506245 DOI: 10.1186/s40478-023-01648-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Accepted: 08/30/2023] [Indexed: 09/20/2023] Open
Abstract
BACKGROUND Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS. METHODS In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO). RESULTS Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients. CONCLUSIONS Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS.
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Affiliation(s)
- Jimmy Beckers
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium.
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium.
| | - Arun Kumar Tharkeshwar
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
- Department of Human Genetics, KU Leuven, Louvain, Belgium
| | - Laura Fumagalli
- Center for Molecular Neurology, Microglia and Inflammation in Neurological Disorders (MIND) Lab, VIB, Antwerp, Belgium
- Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Matilde Contardo
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
| | - Evelien Van Schoor
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
- Laboratory of Neuropathology, Department of Imaging and Pathology, Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
| | - Raheem Fazal
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
| | - Dietmar Rudolf Thal
- Laboratory of Neuropathology, Department of Imaging and Pathology, Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
- Department of Pathology, University Hospitals Leuven, Louvain, Belgium
| | - Siddharthan Chandran
- UK Dementia Research Institute, University of Edinburgh, Edinburgh, EH16 4SB, UK
- Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, UK
| | - Renzo Mancuso
- Center for Molecular Neurology, Microglia and Inflammation in Neurological Disorders (MIND) Lab, VIB, Antwerp, Belgium
- Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium
| | - Ludo Van Den Bosch
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium
| | - Philip Van Damme
- Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Louvain - University of Leuven, Leuven, Belgium.
- Center for Brain and Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium.
- Department of Neurology, University Hospitals Leuven, Louvain, Belgium.
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30
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Li D, Johmura Y, Morimoto S, Doi M, Nakanishi K, Ozawa M, Tsunekawa Y, Inoue-Yamauchi A, Naruse H, Matsukawa T, Takeshita Y, Suzuki N, Aoki M, Nishiyama A, Zeng X, Konishi C, Suzuki N, Nishiyama A, Harris AS, Morita M, Yamaguchi K, Furukawa Y, Nakai K, Tsuji S, Yamazaki S, Yamanashi Y, Shimada S, Okada T, Okano H, Toda T, Nakanishi M. LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits. NATURE AGING 2023; 3:1001-1019. [PMID: 37474791 DOI: 10.1038/s43587-023-00464-4] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/08/2021] [Accepted: 06/29/2023] [Indexed: 07/22/2023]
Abstract
Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2-/- mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.
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Affiliation(s)
- Dan Li
- Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan
| | - Yoshikazu Johmura
- Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan.
- Division of Cancer and Senescence Biology, Cancer Research Institute, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan.
| | - Satoru Morimoto
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Miyuki Doi
- Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Keiko Nakanishi
- Department of Pediatrics, Central Hospital, and Department of Disease Model, Institute for Developmental Research, Aichi Developmental Disability Center, Kasugai, Japan
| | - Manabu Ozawa
- Laboratory of Reproductive Systems Biology, The University of Tokyo, Tokyo, Japan
| | - Yuji Tsunekawa
- Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The University of Tokyo, Tokyo, Japan
| | | | - Hiroya Naruse
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Takashi Matsukawa
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yukio Takeshita
- Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
| | - Naoki Suzuki
- Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Masashi Aoki
- Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Ayumi Nishiyama
- Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Xin Zeng
- Laboratory of Functional Analysis in silico, Human Genome Center, The University of Tokyo, Tokyo, Japan
| | - Chieko Konishi
- Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan
| | - Narumi Suzuki
- Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan
| | - Atsuya Nishiyama
- Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan
| | | | - Mariko Morita
- Division of Clinical Genome Research, The University of Tokyo, Tokyo, Japan
| | - Kiyoshi Yamaguchi
- Division of Clinical Genome Research, The University of Tokyo, Tokyo, Japan
| | - Yoichi Furukawa
- Division of Clinical Genome Research, The University of Tokyo, Tokyo, Japan
| | - Kenta Nakai
- Laboratory of Functional Analysis in silico, Human Genome Center, The University of Tokyo, Tokyo, Japan
| | - Shoji Tsuji
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Satoshi Yamazaki
- Division of Stem Cell Biology, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Laboratory of Stem Cell Therapy, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan
| | - Yuji Yamanashi
- Division of Genetics, The University of Tokyo, Tokyo, Japan
| | - Shoichi Shimada
- Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Takashi Okada
- Division of Molecular and Medical Genetics, Center for Gene and Cell Therapy, The University of Tokyo, Tokyo, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Tatsushi Toda
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Makoto Nakanishi
- Division of Cancer Cell Biology, The University of Tokyo, Tokyo, Japan.
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31
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Onda-Ohto A, Hasegawa-Ogawa M, Matsuno H, Shiraishi T, Bono K, Hiraki H, Kanegae Y, Iguchi Y, Okano HJ. Specific vulnerability of iPSC-derived motor neurons with TDP-43 gene mutation to oxidative stress. Mol Brain 2023; 16:62. [PMID: 37496071 PMCID: PMC10369818 DOI: 10.1186/s13041-023-01050-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Accepted: 07/18/2023] [Indexed: 07/28/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a disease that affects motor neurons and has a poor prognosis. We focused on TAR DNA-binding protein 43 kDa (TDP-43), which is a common component of neuronal inclusions in many ALS patients. To analyze the contribution of TDP-43 mutations to ALS in human cells, we first introduced TDP-43 mutations into healthy human iPSCs using CRISPR/Cas9 gene editing technology, induced the differentiation of these cells into motor and sensory neurons, and analyzed factors that are assumed to be altered in or associated with ALS (cell morphology, TDP-43 localization and aggregate formation, cell death, TDP-43 splicing function, etc.). We aimed to clarify the pathological alterations caused solely by TDP-43 mutation, i.e., the changes in human iPSC-derived neurons with TDP-43 mutation compared with those with the same genetic background except TDP-43 mutation. Oxidative stress induced by hydrogen peroxide administration caused the death of TDP-43 mutant-expressing motor neurons but not in sensory neurons, indicating the specific vulnerability of human iPSC-derived motor neurons with TDP-43 mutation to oxidative stress. In our model, we observed aggregate formation in a small fraction of TDP-43 mutant-expressing motor neurons, suggesting that aggregate formation seems to be related to ALS pathology but not the direct cause of cell death. This study provides basic knowledge for elucidating the pathogenesis of ALS and developing treatments for the disease.
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Affiliation(s)
- Asako Onda-Ohto
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
- Department of Neurology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Minami Hasegawa-Ogawa
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Hiromasa Matsuno
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
- Department of Neurology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Tomotaka Shiraishi
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
- Department of Neurology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Keiko Bono
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
- Department of Neurology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Hiromi Hiraki
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
- Department of Neurology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Yumi Kanegae
- Core Research Facilities, Research Center for Medical Sciences, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Yasuyuki Iguchi
- Department of Neurology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan
| | - Hirotaka James Okano
- Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-Ku, Tokyo, 105-8461, Japan.
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32
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Kume K, Kurashige T, Muguruma K, Morino H, Tada Y, Kikumoto M, Miyamoto T, Akutsu SN, Matsuda Y, Matsuura S, Nakamori M, Nishiyama A, Izumi R, Niihori T, Ogasawara M, Eura N, Kato T, Yokomura M, Nakayama Y, Ito H, Nakamura M, Saito K, Riku Y, Iwasaki Y, Maruyama H, Aoki Y, Nishino I, Izumi Y, Aoki M, Kawakami H. CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis. Am J Hum Genet 2023; 110:1086-1097. [PMID: 37339631 PMCID: PMC10357476 DOI: 10.1016/j.ajhg.2023.05.014] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2023] [Revised: 05/25/2023] [Accepted: 05/25/2023] [Indexed: 06/22/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.
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Affiliation(s)
- Kodai Kume
- Department of Molecular Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Takashi Kurashige
- Department of Neurology, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Hiroshima, Japan
| | - Keiko Muguruma
- Department of iPS Cell Applied Medicine, Graduate School of Medicine, Kansai Medical University, Osaka, Japan
| | - Hiroyuki Morino
- Department of Molecular Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Yui Tada
- Department of Molecular Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Mai Kikumoto
- Department of Molecular Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan; Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, Japan
| | - Tatsuo Miyamoto
- Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Silvia Natsuko Akutsu
- Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Yukiko Matsuda
- Department of Molecular Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Shinya Matsuura
- Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Masahiro Nakamori
- Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, Japan
| | - Ayumi Nishiyama
- Department of Neurology, Tohoku University Graduate School of Medicine, Miyagi, Japan
| | - Rumiko Izumi
- Department of Neurology, Tohoku University Graduate School of Medicine, Miyagi, Japan
| | - Tetsuya Niihori
- Department of Medical Genetics, Tohoku University Graduate School of Medicine, Miyagi, Japan
| | - Masashi Ogasawara
- Department of Neuromuscular Research, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, National Centre Hospital, Tokyo, Japan
| | - Nobuyuki Eura
- Department of Neuromuscular Research, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, National Centre Hospital, Tokyo, Japan
| | - Tamaki Kato
- Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan
| | - Mamoru Yokomura
- Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan
| | - Yoshiaki Nakayama
- Department of Neurology, Wakayama Medical University, Wakayama, Japan
| | - Hidefumi Ito
- Department of Neurology, Wakayama Medical University, Wakayama, Japan
| | | | - Kayoko Saito
- Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan
| | - Yuichi Riku
- Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Nagakute, Japan
| | - Yasushi Iwasaki
- Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Nagakute, Japan
| | - Hirofumi Maruyama
- Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, Japan
| | - Yoko Aoki
- Department of Medical Genetics, Tohoku University Graduate School of Medicine, Miyagi, Japan
| | - Ichizo Nishino
- Department of Neuromuscular Research, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, National Centre Hospital, Tokyo, Japan
| | - Yuishin Izumi
- Department of Neurology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Masashi Aoki
- Department of Neurology, Tohoku University Graduate School of Medicine, Miyagi, Japan
| | - Hideshi Kawakami
- Department of Molecular Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
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33
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Urasawa T, Koizumi T, Kimura K, Ohta Y, Kawasaki N. Quantitative Proteomics for the Development and Manufacturing of Human-Induced Pluripotent Stem Cell-Derived Neural Stem Cells Using Data-Independent Acquisition Mass Spectrometry. J Proteome Res 2023. [PMID: 37097202 DOI: 10.1021/acs.jproteome.2c00841] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/26/2023]
Abstract
Human-induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) have several potential applications in regenerative medicine. A deep understanding of stem cell characteristics is critical for developing appropriate products for use in the clinic. This study aimed to develop approaches for characterizing iPSC-derived NSCs. Data-independent acquisition mass spectrometry (DIA-MS) was used to obtain temporal proteomic profiles of differentiating cells. Principal component analysis of the proteome profiles allowed for the discrimination of cells cultured for different periods. Cells were characterized by Gene Ontology analysis to annotate the upregulated proteins based on their functions. We found that trophoblast glycoprotein (TPBG), a membrane glycoprotein that inhibits the Wnt/β-catenin pathway, was elevated in NSC and that silencing TPBG promoted proliferation rather than neuronal differentiation. Treatment with Wnt/β-catenin pathway activators and inhibitors showed that modulating the Wnt/β-catenin pathway is crucial for differentiation into NSC. These results suggest that the level of TPBG is critical for differentiation into NSC, and TPBG is a potentially critical quality attribute of differentiating cells. In summary, DIA-MS-based proteomics is a promising multi-attribute method for characterizing stem cell-derived products.
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Affiliation(s)
- Takaya Urasawa
- Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 Japan
| | - Takumi Koizumi
- Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 Japan
| | - Kazumasa Kimura
- Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 Japan
| | - Yuki Ohta
- Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 Japan
| | - Nana Kawasaki
- Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 Japan
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34
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Marshall KL, Rajbhandari L, Venkatesan A, Maragakis NJ, Farah MH. Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1 A4V mutation. Sci Rep 2023; 13:5597. [PMID: 37020097 PMCID: PMC10076424 DOI: 10.1038/s41598-023-31720-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Accepted: 03/16/2023] [Indexed: 04/07/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by degeneration of upper and lower motor neurons that leads to muscle weakness, paralysis, and death, but the effects of disease-causing mutations on axonal outgrowth of neurons derived from human induced pluripotent stem cells (iPSC)-derived motor neurons (hiPSC-MN) are poorly understood. The use of hiPSC-MN is a promising tool to develop more relevant models for target identification and drug development in ALS research, but questions remain concerning the effects of distinct disease-causing mutations on axon regeneration. Mutations in superoxide dismutase 1 (SOD1) were the first to be discovered in ALS patients. Here, we investigated the effect of the SOD1A4V mutation on axonal regeneration of hiPSC-MNs, utilizing compartmentalized microfluidic devices, which are powerful tools for studying hiPSC-MN distal axons. Surprisingly, SOD1+/A4V hiPSC-MNs regenerated axons more quickly following axotomy than those expressing the native form of SOD1. Though initial axon regrowth was not significantly different following axotomy, enhanced regeneration was apparent at later time points, indicating an increased rate of outgrowth. This regeneration model could be used to identify factors that enhance the rate of human axon regeneration.
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Affiliation(s)
- Katherine L Marshall
- Neuromuscular Division, Department of Neurology, Johns Hopkins University School of Medicine, The John G. Rangos Sr. Building, Room 239, 855 N. Wolfe Street, Baltimore, MD, 21205, USA
| | - Labchan Rajbhandari
- Neuromuscular Division, Department of Neurology, Johns Hopkins University School of Medicine, The John G. Rangos Sr. Building, Room 239, 855 N. Wolfe Street, Baltimore, MD, 21205, USA
| | - Arun Venkatesan
- Neuromuscular Division, Department of Neurology, Johns Hopkins University School of Medicine, The John G. Rangos Sr. Building, Room 239, 855 N. Wolfe Street, Baltimore, MD, 21205, USA
| | - Nicholas J Maragakis
- Neuromuscular Division, Department of Neurology, Johns Hopkins University School of Medicine, The John G. Rangos Sr. Building, Room 239, 855 N. Wolfe Street, Baltimore, MD, 21205, USA
| | - Mohamed H Farah
- Neuromuscular Division, Department of Neurology, Johns Hopkins University School of Medicine, The John G. Rangos Sr. Building, Room 239, 855 N. Wolfe Street, Baltimore, MD, 21205, USA.
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35
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Lee HJ, Alirzayeva H, Koyuncu S, Rueber A, Noormohammadi A, Vilchez D. Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes. NATURE AGING 2023; 3:546-566. [PMID: 37118550 DOI: 10.1038/s43587-023-00383-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Accepted: 02/17/2023] [Indexed: 04/30/2023]
Abstract
Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective mechanisms to extend longevity in both poikilotherms and homeotherms, a better understanding of cold-induced changes can lead to converging modifiers of pathological protein aggregation. Here, we find that cold temperature (15 °C) selectively induces the trypsin-like activity of the proteasome in Caenorhabditis elegans through PSME-3, the worm orthologue of human PA28γ/PSME3. This proteasome activator is required for cold-induced longevity and ameliorates age-related deficits in protein degradation. Moreover, cold-induced PA28γ/PSME-3 diminishes protein aggregation in C. elegans models of age-related diseases such as Huntington's and amyotrophic lateral sclerosis. Notably, exposure of human cells to moderate cold temperature (36 °C) also activates trypsin-like activity through PA28γ/PSME3, reducing disease-related protein aggregation and neurodegeneration. Together, our findings reveal a beneficial role of cold temperature that crosses evolutionary boundaries with potential implications for multi-disease prevention.
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Affiliation(s)
- Hyun Ju Lee
- Institute for Integrated Stress Response Signaling, Faculty of Medicine, University Hospital Cologne, Cologne, Germany
- Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Hafiza Alirzayeva
- Institute for Integrated Stress Response Signaling, Faculty of Medicine, University Hospital Cologne, Cologne, Germany
- Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Seda Koyuncu
- Institute for Integrated Stress Response Signaling, Faculty of Medicine, University Hospital Cologne, Cologne, Germany
- Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Amirabbas Rueber
- Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - Alireza Noormohammadi
- Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
| | - David Vilchez
- Institute for Integrated Stress Response Signaling, Faculty of Medicine, University Hospital Cologne, Cologne, Germany.
- Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
- Institute for Genetics, University of Cologne, Cologne, Germany.
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
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36
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Du H, Huo Z, Chen Y, Zhao Z, Meng F, Wang X, Liu S, Zhang H, Zhou F, Liu J, Zhang L, Zhou S, Guan Y, Wang X. Induced Pluripotent Stem Cells and Their Applications in Amyotrophic Lateral Sclerosis. Cells 2023; 12:cells12060971. [PMID: 36980310 PMCID: PMC10047679 DOI: 10.3390/cells12060971] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 03/20/2023] [Accepted: 03/20/2023] [Indexed: 03/30/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that results in the loss of motor function in the central nervous system (CNS) and ultimately death. The mechanisms underlying ALS pathogenesis have not yet been fully elucidated, and ALS cannot be treated effectively. Most studies have applied animal or single-gene intervention cell lines as ALS disease models, but they cannot accurately reflect the pathological characteristics of ALS. Induced pluripotent stem cells (iPSCs) can be reprogrammed from somatic cells, possessing the ability to self-renew and differentiate into a variety of cells. iPSCs can be obtained from ALS patients with different genotypes and phenotypes, and the genetic background of the donor cells remains unchanged during reprogramming. iPSCs can differentiate into neurons and glial cells related to ALS. Therefore, iPSCs provide an excellent method to evaluate the impact of diseases on ALS patients. Moreover, patient-derived iPSCs are obtained from their own somatic cells, avoiding ethical concerns and posing only a low risk of immune rejection. The iPSC technology creates new hope for ALS treatment. Here, we review recent studies on iPSCs and their applications in disease modeling, drug screening and cell therapy in ALS, with a particular focus on the potential for ALS treatment.
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Affiliation(s)
- Hongmei Du
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Zijun Huo
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
| | - Yanchun Chen
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Zhenhan Zhao
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
| | - Fandi Meng
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
| | - Xuemei Wang
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
| | - Shiyue Liu
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Haoyun Zhang
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Fenghua Zhou
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
- Department of Pathology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
| | - Jinmeng Liu
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Lingyun Zhang
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Shuanhu Zhou
- Harvard Medical School and Harvard Stem Cell Institute, Harvard University, Boston, MA 02115, USA
| | - Yingjun Guan
- Department of Histology and Embryology, School of Basic Medical Sciences, Weifang Medical University, Weifang 261053, China
- Neurologic Disorders and Regenerative Repair Laboratory, Weifang Medical University, Weifang 261053, China
| | - Xin Wang
- Harvard Medical School and Harvard Stem Cell Institute, Harvard University, Boston, MA 02115, USA
- Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
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KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile. Int J Mol Sci 2023; 24:ijms24065275. [PMID: 36982353 PMCID: PMC10049485 DOI: 10.3390/ijms24065275] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 02/27/2023] [Accepted: 03/05/2023] [Indexed: 03/12/2023] Open
Abstract
Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.
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Jeltema D, Abbott K, Yan N. STING trafficking as a new dimension of immune signaling. J Exp Med 2023; 220:213837. [PMID: 36705629 PMCID: PMC9930166 DOI: 10.1084/jem.20220990] [Citation(s) in RCA: 42] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 01/09/2023] [Accepted: 01/12/2023] [Indexed: 01/28/2023] Open
Abstract
The cGAS-STING pathway is an evolutionarily conserved immune signaling pathway critical for microbial defense. Unlike other innate immune pathways that largely rely on stationary cascades of signaling events, STING is highly mobile in the cell. STING is activated on the ER, but only signals after it arrives on the Golgi, and then it is quickly degraded by the lysosome. Each step of STING trafficking through the secretory pathway is regulated by host factors. Homeostatic STING trafficking via COPI-, COPII-, and clathrin-coated vesicles is important for maintaining baseline tissue and cellular immunity. Aberrant vesicular trafficking or lysosomal dysfunction produces an immune signal through STING, which often leads to tissue pathology in mice and humans. Many trafficking-mediated diseases of STING signaling appear to impact the central nervous system, leading to neurodegeneration. Therefore, STING trafficking introduces a new dimension of immune signaling that likely has broad implications in human disease.
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Affiliation(s)
- Devon Jeltema
- Department of Immunology, UT Southwestern Medical Center, Dallas, TX, USA
| | - Kennady Abbott
- Department of Immunology, UT Southwestern Medical Center, Dallas, TX, USA
| | - Nan Yan
- Department of Immunology, UT Southwestern Medical Center, Dallas, TX, USA,Correspondence to Nan Yan:
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Ito D, Morimoto S, Takahashi S, Okada K, Nakahara J, Okano H. Maiden voyage: induced pluripotent stem cell-based drug screening for amyotrophic lateral sclerosis. Brain 2023; 146:13-19. [PMID: 36004509 DOI: 10.1093/brain/awac306] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Revised: 07/22/2022] [Accepted: 08/09/2022] [Indexed: 01/11/2023] Open
Abstract
Using patient-derived induced pluripotent stem cells, neurodegenerative disease phenotypes have been recapitulated and their pathogenesis analysed leading to significant progress in drug screening. In amyotrophic lateral sclerosis, high-throughput screening using induced pluripotent stem cells-derived motor neurons has identified candidate drugs. Owing to induced pluripotent stem cell-based drug evaluation/screening, three compounds, retigabine, ropinirole and bosutinib, have progressed to clinical trials. Retigabine blocks hyperexcitability and improves survival in amyotrophic lateral sclerosis patient-derived motor neurons. In a randomized clinical trial (n = 65), treatment with retigabine reduced neuronal excitability after 8 weeks. Ropinirole, identified in a high-throughput screening, attenuates pathological phenotypes in patient-derived motor neurons. In a trial limited by a small sample size (n = 20), ropinirole was tolerable and had clinical benefits on function and survival. A phase 1 study of bosutinib has reported safety and tolerability for 12 weeks. Thus, these clinical trials show safety and positive effects and confirm the reliability of stem cell-based drug discovery. This novel strategy leads to reduced costs and time when compared to animal testing and opens new avenues for therapy in intractable diseases.
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Affiliation(s)
- Daisuke Ito
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Satoru Morimoto
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
- Department of Neurology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Shinichi Takahashi
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
- Department of Neurology, Keio University School of Medicine, Tokyo 160-8582, Japan
- Department of Neurology, Saitama Medical University International Medical Center, Saitama 350-1298, Japan
| | - Kensuke Okada
- Department of Neurology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Jin Nakahara
- Department of Neurology, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
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Zhu W, Xu L, Li X, Hu H, Lou S, Liu Y. iPSCs-Derived Neurons and Brain Organoids from Patients. Handb Exp Pharmacol 2023; 281:59-81. [PMID: 37306818 DOI: 10.1007/164_2023_657] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Induced pluripotent stem cells (iPSCs) can be differentiated into specific neurons and brain organoids by adding induction factors and small molecules in vitro, which carry human genetic information and recapitulate the development process of human brain as well as physiological, pathological, and pharmacological characteristics. Hence, iPSC-derived neurons and organoids hold great promise for studying human brain development and related nervous system diseases in vitro, and provide a platform for drug screening. In this chapter, we summarize the development of the differentiation techniques for neurons and brain organoids from iPSCs, and their applications in studying brain disease, drug screening, and transplantation.
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Affiliation(s)
- Wanying Zhu
- School of Pharmacy, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Lei Xu
- School of Pharmacy, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Xinrui Li
- School of Pharmacy, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Hao Hu
- School of Pharmacy, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Shuning Lou
- School of Pharmacy, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Yan Liu
- School of Pharmacy, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
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Abstract
The scientific landscape surrounding amyotrophic lateral sclerosis has shifted immensely with a number of well-defined ALS disease-causing genes, each with related phenotypical and cellular motor neuron processes that have come to light. Yet in spite of decades of research and clinical investigation, there is still no etiology for sporadic amyotrophic lateral sclerosis, and treatment options even for those with well-defined familial syndromes are still limited. This chapter provides a comprehensive review of the genetic basis of amyotrophic lateral sclerosis, highlighting factors that contribute to its heritability and phenotypic manifestations, and an overview of past, present, and upcoming therapeutic strategies.
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Affiliation(s)
- David S Younger
- Department of Clinical Medicine and Neuroscience, CUNY School of Medicine, New York, NY, United States; Department of Medicine, Section of Internal Medicine and Neurology, White Plains Hospital, White Plains, NY, United States.
| | - Robert H Brown
- Department of Neurology, UMass Chan Medical School, Donna M. and Robert J. Manning Chair in Neurosciences and Director in Neurotherapeutics, Worcester, MA, United States
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42
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Akter M, Ding B. Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons. Cells 2022; 11:3796. [PMID: 36497056 PMCID: PMC9737271 DOI: 10.3390/cells11233796] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2022] [Revised: 11/19/2022] [Accepted: 11/24/2022] [Indexed: 11/29/2022] Open
Abstract
Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions.
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Affiliation(s)
| | - Baojin Ding
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA
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Luo HM, Xu J, Huang DX, Chen YQ, Liu YZ, Li YJ, Chen H. Mitochondrial dysfunction of induced pluripotent stem cells-based neurodegenerative disease modeling and therapeutic strategy. Front Cell Dev Biol 2022; 10:1030390. [DOI: 10.3389/fcell.2022.1030390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2022] [Accepted: 10/31/2022] [Indexed: 11/22/2022] Open
Abstract
Neurodegenerative diseases (NDDs) are disorders in which neurons are lost owing to various factors, resulting in a series of dysfunctions. Their rising prevalence and irreversibility have brought physical pain to patients and economic pressure to both individuals and society. However, the pathogenesis of NDDs has not yet been fully elucidated, hampering the use of precise medication. Induced pluripotent stem cell (IPSC) modeling provides a new method for drug discovery, and exploring the early pathological mechanisms including mitochondrial dysfunction, which is not only an early but a prominent pathological feature of NDDs. In this review, we summarize the iPSC modeling approach of Alzheimer’s disease, Parkinson’s disease, and Amyotrophic lateral sclerosis, as well as outline typical mitochondrial dysfunction and recapitulate corresponding therapeutic strategies.
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Robust Generation of Ready-to-Use Cryopreserved Motor Neurons from Human Pluripotent Stem Cells for Disease Modeling. Int J Mol Sci 2022; 23:ijms232113462. [PMID: 36362259 PMCID: PMC9657726 DOI: 10.3390/ijms232113462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Revised: 10/31/2022] [Accepted: 11/02/2022] [Indexed: 11/06/2022] Open
Abstract
Human pluripotent stem cell (hPSC)-derived motor neurons (MNs) act as models for motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy. However, the MN differentiation efficiency and viability following cryopreservation require further development for application in large-scale studies and drug screening. Here, we developed a robust protocol to convert hPSCs into MN cryopreservation stocks (hPSCs were converted into >92% motor neural progenitors and >91% MNs). Near-mature MNs were cryopreserved at a high thawing survival rate and 89% MN marker expression on day 32. Moreover, these MNs exhibited classical electrophysiological properties and neuromuscular junction (NMJ) formation ability within only 4−6 days after thawing. To apply this platform as an MND model, MN stocks were generated from SOD1G85R, SOD1G85G isogenic control, and sporadic ALS hPSC lines. The thawed ALS MNs expressed ALS-specific cytopathies, including SOD1 protein aggregation and TDP-43 redistribution. Thus, a stable and robust protocol was developed to generate ready-to-use cryopreserved MNs without further neuronal maturation processes for application in MND mechanistic studies, NMJ model establishment, and large-scale drug screening.
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45
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Bhargava A, Sandoval Castellanos AM, Shah S, Ning K. An insight into the iPSCs-derived two-dimensional culture and three-dimensional organoid models for neurodegenerative disorders. Interface Focus 2022; 12:20220040. [PMID: 35992771 PMCID: PMC9372641 DOI: 10.1098/rsfs.2022.0040] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2022] [Accepted: 07/18/2022] [Indexed: 12/20/2022] Open
Abstract
The use of induced pluripotent stem cells (iPSCs) is a promising approach when used as models to study neurodegenerative disorders (NDDs) in vitro. iPSCs have been used in in vitro two-dimensional cultures; however, these two-dimensional cultures do not mimic the physiological three-dimensional cellular environment. The use of iPSCs-derived three-dimensional organoids has risen as a powerful alternative to using animal models to study NDDs. These iPSCs-derived three-dimensional organoids can resemble the complexity of the tissue of interest, making it an approachable, cost-effective technique, to study NDDs in an ethical manner. Furthermore, the use of iPSCs-derived organoids will be an important tool to develop new therapeutics and pharmaceutics to treat NDDs. Herein, we will highlight how iPSCs-derived two-dimensional cultures and three-dimensional organoids have been used to study NDDs, as well as the advantages and disadvantages of both techniques.
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Affiliation(s)
- Anushka Bhargava
- Sheffield Institute for Translational Neuroscience, Department of Neuroscience, The University of Sheffield, Sheffield S10 2HQ, UK
| | - Ana M. Sandoval Castellanos
- Sheffield Institute for Translational Neuroscience, Department of Neuroscience, The University of Sheffield, Sheffield S10 2HQ, UK
| | - Sonali Shah
- Sheffield Institute for Translational Neuroscience, Department of Neuroscience, The University of Sheffield, Sheffield S10 2HQ, UK
| | - Ke Ning
- Sheffield Institute for Translational Neuroscience, Department of Neuroscience, The University of Sheffield, Sheffield S10 2HQ, UK
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Khamaysa M, Pradat PF. Status of ALS Treatment, Insights into Therapeutic Challenges and Dilemmas. J Pers Med 2022; 12:1601. [PMID: 36294741 PMCID: PMC9605458 DOI: 10.3390/jpm12101601] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Revised: 09/14/2022] [Accepted: 09/23/2022] [Indexed: 12/18/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is an extremely heterogeneous disease of motor neurons that eventually leads to death. Despite impressive advances in understanding the genetic, molecular, and pathological mechanisms of the disease, the only drug approved to date by both the FDA and EMA is riluzole, with a modest effect on survival. In this opinion view paper, we will discuss how to address some challenges for drug development in ALS at the conceptual, technological, and methodological levels. In addition, socioeconomic and ethical issues related to the legitimate need of patients to benefit quickly from new treatments will also be addressed. In conclusion, this brief review takes a more optimistic view, given the recent approval of two new drugs in some countries and the development of targeted gene therapies.
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Affiliation(s)
- Mohammed Khamaysa
- Laboratoire d’Imagerie Biomédicale, Sorbonne Université, CNRS, INSERM, 75006 Paris, France
| | - Pierre-François Pradat
- Laboratoire d’Imagerie Biomédicale, Sorbonne Université, CNRS, INSERM, 75006 Paris, France
- Centre Référent SLA, Département de Neurologie, AP-HP, Hôpital Pitié-Salpêtrière, 75013 Paris, France
- Northern Ireland Centre for Stratified Medicine, Biomedical Sciences Research Institute, Ulster University, C-TRIC, Altnagelvin Hospital, Derry-Londonderry BT47 6SB, UK
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47
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Amorós MA, Choi ES, Cofré AR, Dokholyan NV, Duzzioni M. Motor neuron-derived induced pluripotent stem cells as a drug screening platform for amyotrophic lateral sclerosis. Front Cell Dev Biol 2022; 10:962881. [PMID: 36105357 PMCID: PMC9467621 DOI: 10.3389/fcell.2022.962881] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Accepted: 07/20/2022] [Indexed: 11/13/2022] Open
Abstract
The development of cell culture models that recapitulate the etiology and features of nervous system diseases is central to the discovery of new drugs and their translation onto therapies. Neuronal tissues are inaccessible due to skeletal constraints and the invasiveness of the procedure to obtain them. Thus, the emergence of induced pluripotent stem cell (iPSC) technology offers the opportunity to model different neuronal pathologies. Our focus centers on iPSCs derived from amyotrophic lateral sclerosis (ALS) patients, whose pathology remains in urgent need of new drugs and treatment. In this sense, we aim to revise the process to obtain motor neurons derived iPSCs (iPSC-MNs) from patients with ALS as a drug screening model, review current 3D-models and offer a perspective on bioinformatics as a powerful tool that can aid in the progress of finding new pharmacological treatments.
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Affiliation(s)
- Mariana A. Amorós
- Laboratory of Pharmacological Innovation, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil
| | - Esther S. Choi
- Department of Pharmacology, Penn State College of Medicine, Hershey, PA, United States
| | - Axel R. Cofré
- Laboratory of Pharmacological Innovation, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil
| | - Nikolay V. Dokholyan
- Department of Pharmacology, Penn State College of Medicine, Hershey, PA, United States
- Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA, United States
| | - Marcelo Duzzioni
- Laboratory of Pharmacological Innovation, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, Alagoas, Brazil
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Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis. Acta Neuropathol 2022; 144:413-435. [PMID: 35778567 PMCID: PMC9381448 DOI: 10.1007/s00401-022-02450-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Revised: 06/02/2022] [Accepted: 06/02/2022] [Indexed: 11/04/2022]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.
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Hua Y, Yoshimochi K, Li J, Takekita K, Shimotsuma M, Li L, Qu X, Zhang J, Sawa Y, Liu L, Miyagawa S. Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells. Stem Cell Res Ther 2022; 13:223. [PMID: 35658933 PMCID: PMC9166585 DOI: 10.1186/s13287-022-02879-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2022] [Accepted: 05/03/2022] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Human-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost. METHODS In this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down's syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2. RESULTS We found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers. CONCLUSIONS Our xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.
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Affiliation(s)
- Ying Hua
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Kenji Yoshimochi
- NACALAI TESQUE, INC. Research and Development Department, Kyoto, 604-0855, Japan
| | - Junjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan.,Division of Cardiovascular Surgery, Department of Design for Tissue Regeneration, Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Kazuhiro Takekita
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Motoshi Shimotsuma
- NACALAI TESQUE, INC. Research and Development Department, Kyoto, 604-0855, Japan
| | - Lingjun Li
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Xiang Qu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | - Jingbo Zhang
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
| | | | - Li Liu
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan. .,Division of Cardiovascular Surgery, Department of Design for Tissue Regeneration, Graduate School of Medicine, Osaka, 565-0871, Japan.
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan.
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TDP-43 regulates cholesterol biosynthesis by inhibiting sterol regulatory element-binding protein 2. Sci Rep 2022; 12:7988. [PMID: 35568729 PMCID: PMC9107471 DOI: 10.1038/s41598-022-12133-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Accepted: 04/22/2022] [Indexed: 12/29/2022] Open
Abstract
Dyslipidemia is considered an essential component of the pathological process of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease. Although TAR DNA Binding Protein 43 kDa (TDP-43) links both familial and sporadic forms of ALS and cytoplasmic aggregates are a hallmark of most cases of ALS, the molecular mechanism and the in vivo relation of ALS dyslipidemia with TDP-43 have been unclear. To analyze the dyslipidemia-related gene expression by TDP-43, we performed expression microarray and RNA deep sequencing (RNA-Seq) using cell lines expressing high levels of TDP-43 and identified 434 significantly altered genes including sterol regulatory element-binding protein 2 (SREBP2), a master regulator of cholesterol homeostasis and its downstream genes. Elevated TDP-43 impaired SREBP2 transcriptional activity, leading to inhibition of cholesterol biosynthesis. The amount of cholesterol was significantly decreased in the spinal cords of TDP-43-overexpressed ALS model mice and in the cerebrospinal fluids of ALS patients. These results suggested that TDP-43 could play an essential role in cholesterol biosynthesis in relation to ALS dyslipidemia.
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